Essentially all organisms use circadian clocks to control daily rhythms in physiology, metabolism and behavior. These clocks are of great clinical importance since their dysfunction can lead to sleep disorders and certain forms of depression. The identification and analysis of clock genes in Drosophila revealed that the circadian timekeeping mechanism is comprised of two interlocked transcriptional feedback loops. CLOCK (CLK)-CYCLE (CYC) heterodimers bind E-box elements to activate feedback regulators period (per), timeless (tim), vrille (vri) and Par domain protein 1e (Pdple), which control circadian transcription. VRI and PDP1e bind V/P-boxes to control rhythms in Clk transcription that peak near dawn, whereas PER and TIM inhibit CLK-CYC activity to control rhythms in E-box dependent clock gene transcription that peak near dusk. Even though CLK-CYC dependent activation and PER-TIM dependent inhibition directly or indirectly control all circadian transcription, how PER-TIM feeds back to inhibit CLK-CYC transcriptional activity is not well understood. We find that CLK-CYC binds E-boxes in vivo only at times when their target genes are transcribed, which differs from the analogous E-box activators in mammals that bind constitutively or bind primarily when target gene transcription is inhibited. This result suggests that PER-TIM functions to inhibit E- box binding by CLK-CYC, thus enabling us to determine which E-boxes are important for CLK-CYC dependent transcription and which genes are directly controlled by CLK-CYC. We also find that CLK phosphorylation is high when CLK-CYC forms a transcriptionally inactive nuclear complex with PER-TIM and DOUBLE-TIME (DBT) kinase, and is low when CLK-CYC binds E-boxes and activates transcription. This, together with results showing CLK phosphorylation is PER dependent and DBT destabilizes CLK, suggests that interactions between CLK and PER-TIM-DBT produce a rhythm in CLK phosphorylation that regulates CLK stability and CLK-CYC transcriptional activity. In this proposal, we will define the molecular mechanisms that control rhythmic transcription by determining how (1) rhythms in CLK-CYC binding activity are regulated, (2) E-boxes contribute to rhythmic transcription within and downstream of the feedback loops, and (3) CLK phosphorylation affects CLK stability and CLK-CYC activity. Since circadian timekeeping mechanismsfrom flies and mammals are well conserved, what we learn here will be relevant to the situation in mammals.